Project Summary/Abstract Malaria continues to be a global health problem, especially in sub-Saharan Africa where the mosquito Anopheles gambiae s.s. serves as the major vector for the protozoan Plasmodium falciparum. A critical component of vector competency is the mosquito immune system. To determine molecular mechanisms of mosquito immunity, vector biologists currently use RNAi as a reverse genetics tool to knockdown genes of interest. While efficient and easily employed, a weakness of RNAi is its lack of tissue specificity. One approach to get around this barrier is the use of transgenic mosquitoes. Unfortunately, transgenic lines are cumbersome to make and maintain, and the number of well characterized tissue-specific promoters is limited. Due to the vast number of attractive genes expressed in multiple tissues, a malleable system for producing tissue-specific gene knockdown would be highly beneficial to the vector biology community. We propose to drive tissue-specific RNAi in the mosquito by combining two components, (1) a pan- tissue tropic, non-pathogenic virus and (2) tissue-specific miRNAs. The virus will infect and deliver short-hairpin RNA molecules to induce RNAi. To restrict the delivery system, endogenous miRNAs will be used to block either virus replication or transgene expression; ultimately resulting in tissue specific gene knockdown. The long-term goal is to decipher gene function in a tissue-specific manner in the mosquito. For this proposal, tissue-specific RNAi proof of principle experiments will focus on the ovary. The specific aims are (1) drive RNAi to every tissue but the ovary, (2) drive RNAi specifically to the ovary, and to obtain a deeper understanding of miRNA expression in mosquitoes, (3) determine miRNA transcriptome for multiple Anopheles gambiae tissues. The health relevance of this proposal is the knowledge gained from understanding gene function at the tissue level in the malaria vector is important for future development of vector control strategies. Once optimized, the proposed somatic gene therapy method for mosquitoes will provide a unique genetic tool of value to the vector biology community as a whole.